Instruments, Proficiency Testing, and Reporting

Instruments

Molecular instruments need the same discipline as any analyzer, plus a few specifics:

• Thermocyclers: CAP requires checking the temperature accuracy of individual wells (or a representative sample) before placing the instrument in service and at least annually. A functional check such as amplification performance can substitute, and for closed systems it’s part of the manufacturer’s preventive maintenance (MOL.49520) ¹.
• Pipettes: calibration checked on a schedule, commonly every 6 to 12 months.
• Centrifuges, heat blocks, freezers, refrigerators: speed and temperature monitored and logged.
• Calibration verification for quantitative assays at least every six months, with new reagent lots, and after major maintenance ¹.
• Preventive maintenance on the manufacturer’s schedule, with records.

Every instrument has a maintenance log; a missing record is treated as maintenance not done.

Proficiency testing

Proficiency testing (PT) sends blind samples from an outside provider (CAP, for most molecular tests) to be tested like patient samples, with results graded against peer labs. PT is usually twice a year for molecular tests ¹. Rules:

• Test PT samples like patient samples, by the staff who normally test, with the usual methods.
• Never send PT samples to another lab or discuss them with other labs before the deadline. PT referral is a serious CLIA violation.
• Investigate and document every unacceptable result, even if the overall event passes.

Most molecular tests aren’t “regulated analytes” listed in CLIA subpart I. For those, and for any test without a compatible CMS-approved PT program, the lab must verify accuracy at least twice a year by another method ². Options include:

• Split samples exchanged with another laboratory.
• Testing previously characterized samples or reference materials.
• Comparison with an alternative method.

Reporting results

A molecular report must say exactly what was tested and found. It typically includes:

• Patient and specimen identifiers, specimen type, collection and receipt dates.
• The test name, method, and genes or targets covered (and what wasn’t covered).
• The result in standard nomenclature, and an interpretation in plain language.
• Limitations: the assay’s limit of detection, variants not detected by the method, the effect of tumor content.
• For lab-developed tests using ASRs, the required statement that the test “was developed and its performance characteristics determined by” the lab and “has not been cleared or approved by the U.S. Food and Drug Administration” ³.

HGVS nomenclature

Sequence variants are written in HGVS format: reference sequence : description ⁴. Without the reference sequence, a description is ambiguous.

Prefixes ⁴:

• g. genomic, c. coding DNA, n. non-coding RNA, r. RNA, m. mitochondrial, p. protein.

Variant types ⁴:

Parentheses around a protein change mean it’s predicted, not directly observed ⁴.

Examples you’ll meet: the sickle variant HBB c.20A>T, p.(Glu7Val) (historically “Glu6Val”, which counted from the second amino acid), and the common cystic fibrosis variant CFTR c.1521_1523del, p.(Phe508del).

What to take from this

Check thermocycler wells before service and annually, verify calibration of quantitative assays every six months and with lot changes, and keep maintenance records. Treat PT samples like patients, never refer them, and investigate every miss. For tests without approved PT, verify accuracy twice a year another way. Report the method, targets, result, interpretation, and limitations, with the ASR disclaimer for LDTs, and write variants in HGVS format with a reference sequence.